A flow cytometry evaluation of anti‐FVIII antibodies: correlation with ELISA and Bethesda assay

Summary. In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme‐linked immunosorbent assay (ELISA) that detects both inhibitory (I‐Ab) and non‐inhibitory (NI‐Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I‐Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII‐m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control‐m). Captured anti‐FVIII antibodies were detected using anti‐human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I‐Ab, <0.5 BU mL^?1); 13 PI (patients with I‐Ab, 1.1–8200 BU mL^?1). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII‐m and control‐m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI‐Ab by FC, and two of them developed high levels of I‐Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 μL of plasma or serum is required especially making it useful for paediatric patients.     

High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post-transfusion antibodies may also arise against non-coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII-induced antibodies by use of an enzyme-linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non-inhibitory antibodies was evaluated in three patients; although half-life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti-FVIII antibodies by ELISA when routinely assessing haemophilia A patients.     

ELISA system for detection of immune responses to FVIII: a study of 246 samples and correlation with the Bethesda assay

Inhibitors of FVIII are usually IgG polyclonal antibodies that develop as alloimmune responses in patients with congenital haemophilia A or as autoimmune responses resulting in acquired haemophilia. Their recognition can be difficult, especially when the titre is low. Furthermore, results from a Bethesda assay often require several days as samples are referred to a specialty laboratory. The aim of this study is to assess the utility of an ELISA system for detecting immune responses to FVIII. A total of 246 plasma samples submitted from 176 individuals with immune responses to FVIII, as verified with the Bethesda assay, and samples from 50 control subjects were tested for the presence of FVIII-specific IgG using an ELISA-based assay. Paired sera from 18 of the patients were also tested by the ELISA. Of the 246 samples that were positive for a FVIII inhibitor by the Bethesda assay, 235 (95.5%) were also positive by ELISA. The regression coefficient, using Log BU was r = 0.82. The correlation data were strengthened when 27 inhibitor samples were diluted further. There was a strong correlation between ELISA results for the 18-paired serum and plasma samples (r = 0.99). There is a strong correlation between the ELISA and Bethesda methods in detecting immune responses to FVIII. The ELISA provides rapid screening that could be available well in advance of confirmation by the Bethesda assay.     

Long‐term anti‐FVIII antibody response in Bethesda‐negative haemophilia A patients receiving continuous replacement therapy

It has previously been shown that patients with haemophilia A may develop non‐neutralizing anti‐factor VIII (FVIII) antibodies (NNA) that escape detection by the Bethesda assay, but are detected using immune‐based assays. We and others found NNAs to be directed not only towards non‐functional parts of the protein, but towards all regions of the FVIII protein. We also showed a heterogeneous antibody response towards different FVIII products. However, the clinical relevance and the natural history of NNA remain unclear. Therefore, we followed a cohort of unrelated subjects with haemophilia A for 4 years with the goal of exploring the long‐term development of NNA using an enzyme‐linked immunosorbent assay (ELISA). Ten of 78 subjects (12·8%) exhibited an immune response that was transient and heterogeneous, and none of the subjects developed an FVIII inhibitor. The result of the ELISA was examined in relation to clinical variables and no significant associations between a positive ELISA and age, F8 mutation, port‐à‐cath implantation and HCV infection were shown. Interestingly, patients with NNA had significantly fewer bleeding episodes (P = 0·048) compared with NNA‐negative subjects. The results indicate that the immune response to FVIII products within an individual may vary over time. However, the clinical impact of NNA remains unclear.     

Clinical audit of antiphospholipid antibody testing in tertiary practice: towards improved relevance in thrombophilia investigations

Background: The antiphospholipid syndrome (APS) is an autoimmune condition characterised by vascular thromboses and/or pregnancy morbidity. Diagnosis of APS typically requires laboratory evidence of antiphospholipid antibodies (aPL). Depending on their clinical presentation, affected individuals might be seen by a variety of clinical specialities. Aim: To evaluate clinical ordering patterns for aPL/APS at a tertiary level public facility. Methods: We performed an audit of internal clinical requests for aPL tests at our institution for a 6‐month period. Results: We identified a wide variety of clinical ordering background for aPL, of predominantly obstetric (72/268; 26.9%) or thrombophilic (78/268; 29.1%) patients. Only 11/268 samples (4.1%) were positive for lupus anticoagulant (LA) and 14/268 (5.2%) were positive for anticardiolipin antibody (aCL). The percentage of aCL positivity in the LA‐positive group was 46% (5/11). None of the 72 obstetric patients tested was identified to have aPL. Of the 11 LA‐positive patients, the reasons identified for testing comprised: prolonged Activated Partial Thromboplastin Time (assay) (n= 3), thrombosis (n= 3), APS (n= 2), systemic lupus erythematosus (n= 2), vasculitis (n= 1). Conclusion: We determined a wide variety of clinical ordering background for aPL at a tertiary level institution, with an overall low rate (<10%) of aPL positivity among a hospital population of predominantly obstetric or thrombophilic patients. That no positive obstetric aPL cases were identified suggests local clinical ordering guidelines may need review, as also potentially practised at other institutions. We also observed a moderate rate (46%) of coincidence of aCL and LA, in agreement with guidelines indicating that multiple tests are required to identify APS.     

Anticuerpos antifosfatidilserina/protrombina en pacientes con poliarteritis nodosa

IntroductionAnti-phosphatidylserine/prothrombin (aPS/PT) antibodies have been described in cutaneous Polyarteritis Nodosa (PAN) in association with specific manifestations.ObjectivesTo determine aPS/PT antibodies in patients with PAN and its correlation with clinical manifestations.MethodsCross-sectional comparative study including PAN patients and 20 controls (10 Microscopic Polyangiitis [MPA] and 10 Behçet's disease [BD]). Clinical and demographic variables, treatment, serological markers, prognosis, activity and damage indexes were evaluated. aPS/PT, anti-cardiolipin (aCL), anti-beta 2 glycoprotein 1 (anti-B2GP1) antibodies, and lupus anticoagulant (LA) were determined.ResultsFourteen patients with PAN were included, 11 (79%) women, with disease duration of 207 months, and mostly inactive disease. Only one patient with PAN and one with BD were positive for aPS/PT IgG. LA was the most frequent antibody identified. One patient with MPA and one with BD were positive for aCL IgM; one with MPA for anti-B2GP1 IgG, and one with PAN for anti-B2GP1 IgM.ConclusionsaPS/PT antibodies are not frequent in patients with longstanding inactive PAN.     

Immunoglobulin isotypes and functional anti‐FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice

Summary. Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti‐factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16‐disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T‐ and B‐cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti‐FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII‐treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII‐treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro‐environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain‐dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.     

Antibodies to factor VIII in plasma of patients with hemophilia A and normal subjects

 Non-neutralizing factor VIII (FVIII) antibodies (FVIII-Ab) in hemophilia A may be associated with an abnormal clinical response to FVIII concentrates. Patients with FVIII inhibitors may develop noncoagulation FVIII-Ab after the induction of immunotolerance. Natural FVIII-Ab may be detected in the plasma of some healthy subjects. The aim of this study was to analyze the presence of FVIII-Ab in the plasma of 53 normal blood donors and 124 patients with hemophilia A (18 patients had a previous history of FVIII inhibitor, but only 12 had inhibitor at the moment this study was performed). FVIIII inhibitor was measured using the Bethesda method. FVIII-Ab were analyzed by a specific ELISA assay using purified FVIII from a monoclonal concentrate and a standard plasma containing 26 Bethesda units (BU) of FVIII inhibitor. Purified FVIII was used to coat wells of a microtiter plate and was incubated with dilutions of plasma to be tested. Bound human IgG FVIII-Ab were detected by incubation with polyclonal sheep anti.human IgG alkaline phosphatase conjugate, and the OD₄₀₅ was quantitated. A linear fit was obtained (by plotting FVIII-Ab positivity [OD 405nm] versus BU titer) when serial dilutions of this standard inhibitor plasma, containing titers of 0.5 BU or higher, were used. Four different levels of FVIII-Ab positivity [OD 405nm] were distinguished in this assay: Negative levels (–) were obtained with dilutions of the standard inhibitor containing <0.5 BU. Mild levels (+) were obtained with dilutions of 0.5–5 BU. Moderate levels (++) were obtained for dilutions ranging from 5–25 BU. Maximum positivity (+++) was obtained for dilutions of titers > 25 BU. FVIII-Ab positivity was detected in eight of the normal subjects (15%): three were found to be moderately positive (++) and five mildly positive (+). No inhibitory activity was detectable when whole plasma was used. All the hemophilic patients with a presence of FVIII inhibitor at the time of the study were found to be positive for FVIII-Ab. In addition, the level of positivity correlated with the corresponding BU. Four of the six patients who had a history of inhibitor were negative and two positive. Twenty additional patients (16.12%) in whom no inhibitory activity was detected were found to be positive for FVIII-Ab: 16 + and four ++. The mean age of patients with FVIII-Ab positivity was significantly higher than that of patients of the FVIII-Ab negative group (p<0.005). In conclusion, FVIII-Ab positivity in patients with hemophilia A was 17.7% higher than the level of positivity detected by an inhibitory assay. We propose that this method for FVIII-Ab analysis could be used for patients with hemophilia A, at least to complement the functional inhibitor assay. FVIII recovery or half-life should be assessed in patients who test positive for FVIII-Ab and who show no evidence of inhibitor. Received: 31 July 1995 / Accepted: 25 January 1996     

Antiphospholipid antibodies and hypertension

Hypertension is a common manifestation of antiphospholipid syndrome (APS). Antiphospholipid antibodies (aPL) have been described in patients with hypertension secondary to renal artery stenosis (RAS). Twenty-six patients with RAS and 25 patients with severe essential hypertension (diastolic blood pressure > 110 mmHg or > or = 3 hypertensive drugs) were studied and compared to 61 age- and sex-matched healthy subjects. Serum samples were tested for lupus anticoagulant (LA), anticardiolipin (aCL) IgG and IgM, antiprothrombin (aPT) IgG and IgM, anti-beta2glycoprotein 1 (abeta2GP1) IgG and IgM. aPL were negative in all patients with RAS. Two patients with essential hypertension had positive aPL (8%) (LA in one patient confirmed in a second assay and abeta2GP1-IgG in the other patient confirmed one year later together with aCL IgG positivity). Among healthy subjects, one case (1.6%) was found to be positive for LA, aCL IgM, abeta2GP1 IgM, aPT IgG, aPT IgM. In conclusion, the association between RAS and aPL seems to be casual rather than an expression of an elective thrombotic localization ofAPS. The positive finding of aPL in 8% of patients with essential hypertension, a frequency higher than that of the control population, deserves further studies in larger series to better explore the relationship between aPL and hypertension.     

Anti-cardiolipin and anti-beta2 glycoprotein I antibodies in Indian patients with systemic lupus erythematosus: association with the presence of seizures

The aim of this study was to examine whether the clinical features of antiphospholipid antibody syndrome are associated with anti-cardiolipin and anti-beta2 glycoprotein I antibodies in Indian patients with SLE. Seventy-six patients (71 females), who fulfilled 1982 ACR criteria for SLE, were prospectively studied for the clinical features of antiphospholipid antibody syndrome (APS), and their sera were analysed for the presence of IgG/IgM/IgA anti-cardiolipin antibodies (aCL) by an in-house ELISA and, in 65 of them, for the presence of IgG anti-beta2 glycoprotein I antibodies (anti-beta2 GPI) by a commercial kit. Thirty-nine (51%) patients were positive for aCL, all of which were positive for IgG aCL, either alone (79.6%) or along with IgM and/or IgA. Twenty-seven (69.3%) out of 39 aCL-positive and seven (26.9%) out of 26 aCL-negative sera were positive for IgG antibodies to beta2 GPI. There was a significant correlation (r = 0.66, P < 0.05) between the levels of aCL and anti-beta2 GPI antibodies. Forty-one patients had features of definite or suggestive APS. Thrombocytopenia, recurrent pregnancy loss and CNS manifestations (seizures eight, infarct one) were seen in 20, 13 and nine patients, respectively. Thrombosis of the peripheral vessels was seen in only one patient. Only the presence of seizures was significantly associated with the presence of aCL and anti-beta2 GPI antibodies (P < 0.05). The characteristic association of definite APS (recurrent pregnancy loss and arterial/venous thrombosis) was lacking.     

Clinical significance of antiphospholipid antibodies in Indian scleroderma patients

In patients with systemic sclerosis (SSc), antiphospholipid antibodies (aPL) have been reported to be associated with more severe manifestations including digital infarct, gangrene and pulmonary hypertension. But these findings are not consistent in all studies; moreover, there are no data available from Indian subcontinent. The objective of this study is to assess the prevalence of antiphospholipid antibodies in Indian SSc patients and correlate them with clinical and immunological features. Seventy-two patients were recruited prospectively from rheumatology clinic from 2002 to 2006. Their medical records were reviewed. Anticardiolipin antibodies (IgG, IgM) by ELISA and lupus anticoagulant (LA) were tested in standardized pattern and repeated after 6 weeks. Anti-β2 glycoprotein-I antibodies were done in patients who had aPL antibodies. Nineteen patients had diffuse cutaneous SSc and 53 had limited disease. Seven patients (9.7%) were positive for aPL antibodies in their sera. Only one patient had clinical features of antiphospholipid antibody syndrome and manifested with recurrent abortions and deep vein thrombosis. She was positive for aCL, LA and anti-β2 glycoprotein-I antibodies. Four patients were only aCL (IgG) positive in moderate titers and one each had only aCL (IgM) and LAC positivity. None of the clinical parameters showed an association with aPL antibody.     

Heparin-induced thrombocytopenia: evaluation of IgG and IgGAM ELISA assays

Background: Heparin‐induced thrombocytopenia (HIT) is a rare complication of heparin therapy resulting from antibody production to platelet factor 4 and heparin complexes (H‐PF4). Methods: We have evaluated four enzyme‐linked immunosorbent assay (ELISA)‐based screening tests to identify the best assay(s) with the highest specificity but without underdiagnosis of HIT. As functional assays are difficult to perform, ELISAs are useful to provide clinicians with a timely answer. Over a 10‐month period, all samples (N = 107) referred to our laboratory were tested for HIT antibodies using four commercially available ELISA kits, two detecting IgG/A/M anti‐H‐PF4 antibodies and the other two IgG specific. Results: Twenty‐eight samples were positive by at least one assay; IgGAM ELISAs were found to be more sensitive with 24 samples positive by Asserachrom IgGAM and 23 by Zymutest IgGAM. Only 18 samples were positive by GTI‐PF4‐IgG and Zymutest IgG. The gold standard serotonin release assay (SRA) was used as a confirmation assay, and 11/28 samples tested positive. All these SRA‐positive samples were positive by all four assays. None of the IgGAM‐only‐positive samples was found to be positive by SRA suggesting a better specificity for the IgG‐only assays. Conclusion: Our data strongly support the use of IgG‐only assays for the detection of HIT antibodies.     

Evaluation of three commercial ELISA kits for anticardiolipin and anti‐beta2‐glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome

Introduction: The laboratory criteria of the antiphospholipid syndrome (APS) include lupus anticoagulant (LAC), anticardiolipin antibodies (aCL) and anti‐β2glycoprotein I antibodies (aβ2GPI) IgG or IgM. Methods: We evaluated three commercial ELISAs for aCL and aβ2GPI IgG and IgM: Asserachrom^® (‘Stago’), Bio‐Rad (‘BR’) and the Bindazyme? (the Binding Site, ‘BS’). Results: Results of all assays and of LAC were correlated with the clinical background (n = 228). Sensitivity for Stago/BS/BR aCL IgG was 14%/15%/18%, for aCL IgM 1%/5%/4%, for aβ2GPI IgG 9%/10%/17% and for aβ2GPI IgM 4%/4%/3%. The specificity for Stago/BS/BR for all assays ranged from 86% to 98%. The positive predictive value (PPV) for Stago/BS/BR aCL IgG was 46%/52%/40%, for aCL IgM 8%/36%/19%, for aβ2GPI IgG 70%/67%/45% and for aβ2GPI IgM 23%/23%/20%. Combining LAC with aCL and aβ2GPI antibodies increased the sensitivity (Stago/BS/BR IgG: 26%/27%/31%, IgM: 22%/21%/26%) and PPV (Stago/BS/BR IgG: 41%/46%/36%, IgM: 34%/40%/36%). Comparing the diagnostic power of the tests, only Stago/BS aβ2GPI IgG had a Chi‐square P‐value lower than 0.05. The combination of LAC and IgG ELISAs of BS resulted in the lowest P‐value (0.098) compared to the other combinations. Conclusion: All evaluated ELISAs are a practical tool in the laboratory diagnosis of APS. The diagnostic performance shows slight differences between the ELISAs from the different manufacturers.     

Functional characteristics of N8, a new recombinant FVIII

Summary. The aim of this study was to evaluate the in vitro function of the new recombinant factor VIII (FVIII) compound, N8. The specific activity of N8 as measured in a FVIII:C one‐stage clot assay was 9300 ± 400 IU mg^?1 based on the analysis of seven individual batches. The ratio between the FVIII:C activity measured in clot and chromogenic assays was 1.00 (95% confidence interval 0.97–1.03). N8 bound to von Willebrand factor with K_d values of 0.2 nm when measured by ELISA and by surface plasmon resonance. FVIIIa cofactor activity was determined from the kinetic parameters of factor IXa‐catalysed factor X (FX) activation. The rate of activation of N8 by thrombin as well as K_m and k_cat for FX activation was in the same range as those observed for Advate^®. The rate of activated protein C (APC)‐catalysed inactivation was similar for activated N8 and Advate^®. N8 improved thrombin generation in a dose‐dependent manner and induced similar rates of thrombin generation as Advate^® and the plasma‐derived FVIII product Haemate^®. Using thromboelastography (TEG^®), N8 was shown to improve the clot formation and clot stability in whole blood from haemophilia A patients. Comparable potency and efficacy of N8 and Advate^® was found based on TEG^® parameters. Finally, similar binding profiles to immobilized lipoprotein receptor‐related protein (LRP) of N8 and Advate^® were observed. The study demonstrated that N8 is fully functional in a variety of assays measuring FVIII activity. No functional differences were found between N8 and comparator compounds.     

Antibody formation and specificity in Bethesda‐negative brother pairs with haemophilia A

Antibodies directed towards non‐neutralizing epitopes on the factor VIII protein (FVIII) may be detected in patients with haemophilia A. We evaluated the prevalence of non‐neutralizing antibodies, in 201 inhibitor‐negative brother pairs with severe haemophilia A, enrolled in the Malmö International Brother Study and the Haemophilia Inhibitor Genetics Study. To evaluate binding specificity of the antibodies, ELISA plates were coated with two recombinant full‐length (FL) FVIII‐products and one recombinant B‐domain‐deleted (BDD) product. Seventy‐nine patients (39.3%) had a history of positive inhibitor titre measured by Bethesda assay, and FVIII antibodies were detected in 20 of them (25.3%). Additional 23 samples from subjects without a history of FVIII inhibitors were ELISA‐positive corresponding to a frequency of non‐neutralizing antibodies of 18.9%. The antibody response towards the different FVIII products was heterogenous, and was raised not only towards the non‐functional B‐domain but also towards both FL‐rFVIII and BDD‐rFVIII. In patients considered successfully treated with immune tolerance induction, 25.4% had remaining FVIII antibodies. The number of families with an antibody response in all siblings was increased when the total antibody response was taken into account, further supporting the concept of a genetic predisposition of the immune response. Further studies and careful monitoring over time are required to appreciate the immune response on the risk of inhibitor development or recurrence in the future.     

Anti β2 glycoprotein I antibodies: detection and association with thrombosis

Summary. It has been demonstrated that antiphospholipid antibodies (aPL) recognize epitopes formed by anionic phospholipids and protein cofactors. β₂ glycoprotein I (β₂GPI) is accepted as the cofactor of anticardiolipin antibodies (aCL). In the present study we explored the presence and clinical associations of anti β₂GPI antibodies of IgG isotype (aβ₂GPI-IgG), measured by ELISA. We studied sera from 169 patients with aCL and/or lupus anticoagulant (LA), including 52 patients with systemic lupus erythematosus and 49 with primary antiphospholipid syndrome (PAPS). We found 31.9% positive sera for aβGPI-IgG in the whole population and 48.6% in the aCL-IgG(+) group. There was a good correlation between the titre of aCL-IgG and the optical density for aβGPI-IgG ( r = 0.69, P <0.01). The presence of aβ₂GPI-IgG was associated with the presence of aCL-IgG ( P <0.0001) and LA ( P <0.0005). However, none of 23 LA (+) patients without aCL had aβ₂GPI-IgG.
We found a statistically significant association between the presence of aβ₂GPI-IgG and a history of venous thromboembolism (VTE) in our patients ( P <0.005). This association was observed in PAPS ( P <0.05) but not in secondary antiphospholipid syndrome (SAPS).
Our study confirms that some aPL(+) sera react with β₂GPI in special experimental conditions. In addition, the presence of these antibodies is associated with a history of VTE.     

Estimation of anticardiolipin antibodies, anti-beta2 glycoprotein I antibodies and lupus anticoagulant in a prospective longitudinal study of children with juvenile idiopathic arthritis

OBJECTIVE: Anticardiolipin antibodies (aCL) have been frequently detected in juvenile idiopathic arthritis (JIA), but have not been associated with disease activity or clinical features of the antiphospholipid syndrome (APS). Our aim was to determine aCL and anti-beta2 glycoprotein I (anti-beta2GPI) antibody levels and lupus anticoagulant (LA) in serial samples from children with JIA and to investigate the clinical significance of these antibodies. METHODS: The values of aCL, anti-beta2GPI and LA were prospectively followed in 28 children with JIA from disease onset. aCL and anti-beta2GPI were assayed by an ELISA method. Two monoclonal beta2GPI-dependent aCL (HCAL and EY2C9) were used as calibrators. LA was determined by a modified dilute Russell viper venom time test. RESULTS: Thirteen (46.4%) children with JIA were already positive for aCL at their first referral to our center. During the follow-up, the frequency of aCL decreased from 46.4% to 28.6%; however, it remained significantly higher compared with healthy children. In contrast, for anti-beta2GPI the difference in the frequency between the children with JIA and healthy children was not statistically significant. Serial determination of aPL levels in JIA patients revealed frequent fluctuations. Positive aCL persisted over time in 6 (21.4%) children with JIA, 6 (21.4%) children were initially positive for aCL, but became later negative, and 3 (10.7%) children were initially negative for aCL and became later positive. Persistently positive anti-beta2GPI were observed during the follow-up only in one patient, while none of the patients was persistently positive for LA. No association between aCL, anti-beta2GPI or LA and disease activity could be established. No patient with positive aCL, anti-beta2GPI or LA showed any clinical feature of APS. CONCLUSION: The discrepancy between the presence of aCL and anti-beta2GPI might indicate that the production of aCL in JIA is associated with an infectious trigger. Furthermore, the low frequency of anti-beta2GPI and LA could explain the limited prothrombotic potential of aPL observed in JIA. However, we found a distinct group of JIA patients with persistently positive aCL, the clinical implications of which are at the present time unknown.     

IgA anticardiolipin and anti-beta2-glycoprotein I are the most prevalent isotypes in African American patients with systemic lupus erythematosus.

BACKGROUND: Ethnicity plays a role in the prevalence, isotype distribution, and clinical significance of anticardiolipin (aCL) and anti-beta2-glycoprotein I (anti-beta2-GPI) antibodies in patients with systemic lupus erythematosus (SLE). Few studies have been done in the African American population. METHODS: Serum samples from 100 African American patients with SLE were tested for IgG, IgM, and IgA aCL and anti-beta2-GPI antibodies by enzyme-linked immunosorbent assay (ELISA). Computerized clinical data on these patients were reviewed with a specific focus on clinical manifestations of antiphospholipid syndrome (APS). RESULTS: Positivity for at least one isotype of aCL antibodies was found in 33% of the patients, whereas 28% were positive for at least one isotype of anti-beta2-GPI antibodies. IgA was the most prevalent isotype for both antibodies; 24% of the patients in the aCL ELISA and 19% in the anti-beta2-GPI ELISA were positive for IgA. Positivity for both aCL and anti-beta2-GPI in the same patient was seen more frequently with the IgA isotype. Fewer than half of the patients positive for aCL antibodies had medium-to-high levels of antibodies. A few patients had presented thrombotic manifestations, and these patients were positive for aCL (P = 0.01) and anti-beta2-GPI antibodies (P = 0.02). No other manifestations of APS could be significantly correlated with the presence of these antibodies. CONCLUSIONS: Our results show that IgA is the most prevalent isotype among the African American patients with SLE studied. The predominance of the IgA isotype and the low prevalence of medium-to-high levels of aCL antibodies may account for the low frequency of clinical manifestations of APS in these patients.     

Comparison between the standard anticardiolipin antibody test and a new phospholipid test in patients with connective tissue diseases

OBJECTIVE: Antiphospholipid (aPL) antibodies are present in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid antibody syndrome (APS) and are associated with recurrent thromboses, thrombocytopenia, and pregnancy losses. The presence of aPL antibodies is routinely tested using a standardized ELISA that utilizes cardiolipin as antigen (aCL ELISA). This test, although sensitive, is frequently positive in patients with nonrelated autoimmune disorders and some infectious diseases, making the test less specific. Thus there is a need for more specific tests for aCL with equivalent sensitivity to the standard assay. We evaluated the diagnostic utility of a new aPL antibody test kit with a unique phospholipid mixture designed to be more specific than the standard anticardiolipin ELISA. METHODS: aPL antibodies (IgG, IgM) were measured by both a standard ELISA and a new ELISA kit (APhL ELISA Kit, Louisville APL Diagnostics, Inc., Louisville, KY, USA) in the baseline serum from patients enrolled in a 5 year inception cohort, prospective study of early rheumatoid diseases: rheumatoid arthritis (N = 70), SLE (70), scleroderma (45), inflammatory myositis (36), and early undifferentiated connective tissue disease (CTD) (165). Diagnosis was based on standardized criteria and determined at the last study visit. A nested group of patients with Sjogren's syndrome (44) was also defined. Serum from 200 blood donors (BD) served as controls. Patients with known APS (33) and antinuclear cytoplasmic antibody positive renal vasculitis (52) were also studied. Laboratory personnel were blinded to sample diagnostic group. RESULTS: The kit was 90.9% sensitive for detecting APS. Seven patients missed by the kit all had standard aCL values < 40 PL units. Assuming controls do not have APS, the kit was 99.5% specific vs 96.0% for the standard assay. For the patients with CTD, the kit never detected a patient that was not also detected by the standard aCL assay. CONCLUSION: The APhL ELISA Kit appears to be more specific than the standard aCL ELISA without adding potential false positive results. The new test may be useful for followup study for patients found to be aCL positive by standard assays to increase specificity for aCL screening.     

Antiphospholipid antibodies in HIV-positive patients

Antiphospholipid (aPL) antibodies classically have been associated with thrombotic phenomena and abortion in patients with autoimmune diseases. The objective of the present work was to evaluate the frequency of such antibodies in patients infected with HIV and study its association with the presence of clinical manifestations of antiphospholipid syndrome (APS). Using a transversal study, a population of patients diagnosed with HIV, identified through an enzyme-linked immunosorbent assay (ELISA) test and confirmed by Western blotting, aged above 17 years old, was investigated. Through a standard questionnaire, the presence of APS manifestations was investigated, as well as the frequency of rheumatic manifestations. Antibodies against beta2 glycoprotein I (anti-beta2 GPI) and anticardiolipin (aCL) IgA, IgG, and IgM were investigated by the ELISA method using commercial kits (QUANTA Lite, INOVA Diagnostics). Ninety patients were studied, 47 (52.2%) male and 43 (47.8%) female. Clinical manifestations of APS were detected in 12 patients (13.3%) of the studied population, whereas arthralgia was the most common rheumatic manifestation (38.9%). Of the 90 patients, 40 (44.4%) were reactive for at least one type of aPL antibody (aCL and/or anti-beta2 GPI). The frequency of aCL was 17.8%, from which 15 (16.7%) had aCL IgG, 3 (3.3%) IgM, and 1 (1.1%) IgA. The frequency of the anti-beta2 GPI antibody was 33.3%, from which 29 (32.2%) were positive for isotype IgA, 4 (4.4%) isotype IgM, and 1 (1.1%) isotype IgG. No association was observed between immunoreactivity for aPL antibodies in general or each isotype in particular and the presence of APS manifestation. In the present study, it was possible to observe a relatively high frequency of aPL antibodies, particularly for isotype IgA anti-beta2 GPI in HIV. However, there was no association to APS manifestations, suggesting that such antibodies had no etiopathogenic role in these complications in patients with such retroviral infection.